Novel levanbiohydrolase and method of use thereof

Abstract

The present invention relates to a polypeptide comprising an amino acid sequence having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 % of the activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The invention further relates to the use of such a polypeptide for the biotechnological production of levanbiose, and to a method for the production of levanbiose using such a polypeptide.

Description

[0001] NOVEL LEVANBIOHYDROLASE AND METHOD OF USE THEREOF

[0002] TECHNICAL FIELD

[0003] The present invention relates to a polypeptide comprising an amino acid sequence having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, obtainable by N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 % of the activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The invention further relates to the use of such a polypeptide for the biotechnological production of levanbiose, and to a method for the production of levanbiose using such a polypeptide.

[0004] BACKGROUND OF THE INVENTION

[0005] A large amount of epidemiological data shows that excessive sugar consumption affects human health beyond the mere intake of calories. Experts have come to believe that high-calorie sugars induce numerous diseases associated with metabolic syndrome. These diseases include obesity, high blood pressure, an increase in triglyceride content due to increased fat synthesis in the liver, insulin resistance, diabetes and ageing processes caused by the non-enzymatic binding of fructose to lipids, proteins and DNA.

[0006] A widespread measure to reduce global sugar consumption is the substitution of high-calorie sugar with low-calorie or calorie-free sugar substitutes. The use of this class of substances, which is divided into sweeteners and sugar substitutes, in finished products and especially soft drinks is steadily increasing. Recently, however, there has been increasing evidence that artificial sweeteners such as saccharin, sucralose and aspartame have a negative effect on the human microbiota and increase the pathogenic properties of Escherichia coli and Enterococcus faecalis. In addition, the synthetic structure of many sweeteners prevents their degradation by natural decomposers or water treatment plants, whereby synthetic sweeteners increasingly accumulate in aquatic systems and disrupt the circular economy principle on which the bioeconomy is based.

[0007] In the context of the rapid rise in obesity and sugar-associated diseases, there is a need for sustainable sugar substitutes that provide consumers with a satisfying taste experience without negatively impacting metabolic and economic processes and the intestinal microbiota, preferably even promoting overall health.

[0008] In addition to the general trend for sugar substitution in food products and beverages another ongoing trend of consumers or a least a substantial share of consumers is the preference for foods and beverages that are considered to be healthier in that they have prebiotic or probiotic properties. There is thus also generally a need in the art for food additives that have prebiotic and / or probiotic properties.

[0009] It has been found that, compared to sucrose (table sugar), levanbiose has a sweetening power of 0.55 and is thus suitable as a sucrose substitute. Levanbiose is further known to promote the growth of probiotic bacteria, such as bifidobacteria. Levanbiose (fructofuranosyl-(2->6)-Q-p-D-fructose) is a disaccharide obtainable via hydrolysis of the fructan levan.

[0010] While various levan-hydrolyzing enzymes are known to produce levanbiose, examples of which include the levanbiohydrolases of Streptomyces sp. 7-3, Pseudomonas sp. 43, Arthrobacter sp. No. 51A, Streptomyces exfoliates F3-2, and Microbacterium laevaniformans ATCC 15953, most suffer from low specificity towards levanbiose synthesis and incomplete substrate turnover. The native LevM from Microbacterium laevaniformans ATCC 15953, for example, generates substantial by-products such as levantriose and free fructose, and leaves a considerable fraction of levan unconverted even after prolonged incubation (E.K. Song, H. Kim, H.K. Sung, J. Cha, Cloning and characterization of a levanbiohydrolase from Microbacterium laevaniformans ATCC 15953, Gene 291 (2002) 45-55). Thus, the levan-hydrolyzing enzymes known in the art are generally unsuitable for use in the economical production of levanbiose, the reasons for which being on the one hand generation of considerable amounts of side products, which overall complicates and hampers purification of levanbiose, and / or insufficient levanbiose-generating activity on the other hand.

[0011] Therefore, there is also a need in the art for a levanbiose-producing enzyme, which mitigates one or more of the above shortcomings of known levanbiose-producing enzymes and that allows for the economical production of levanbiose.

[0012] SUMMARY OF THE INVENTION

[0013] The inventors of the present invention have developed an improved levanbiohydrolase variant, said variant being, relative to its wildtype parent, genetically optimized in terms of levanbiose-producing activity as well as in terms of expression rates of said enzyme in the respective host cell.

[0014] In a first aspect, the present invention thus provides for a polypeptide comprising an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, obtainable by N-terminal and, optionally, C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the activity, particularly levanbiose-generating activity, of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0015] In some embodiments, the polypeptide is characterized in that the N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0016] Alternatively or additionally, in some embodiments, the C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, most preferably by amino acids 553-620 of SEQ ID NO:2. Alternatively or additionally, in some embodiments, the polypeptide comprises an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

[0017] Alternatively or additionally, in some embodiments, the polypeptide is an isolated polypeptide.

[0018] In a further aspect, the present invention provides a nucleic acid encoding the polypeptide of the present invention, as herein described.

[0019] In another aspect, the present invention provides a vector comprising a nucleic acid molecule according to the present invention, wherein the vector preferably is a plasmid.

[0020] In yet another aspect, the present invention provides a host cell comprising a nucleic acid molecule according to the present invention or a vector according to the present invention, the host cell preferably being a prokaryotic host cell.

[0021] In some embodiments, said host cell is selected from the group consisting of E. coli, in particular E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coli XL , E. coli NEB5-a, E. coli DH5a, E. coli DH1 , E. coli DM1 , E. coli HB101 , E. coli JmlOI-110, E. coli Rosetta(DE3)pLysS, E. co / / SURE, E. coli TOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue.

[0022] In another aspect, the present invention provides a method for the production of a polypeptide according to the present invention, said method comprising

[0023] (1) cultivating a host cell according to the present invention under conditions that allow the expression of the polypeptide according to the present invention; and optionally

[0024] (2) isolating the expressed polypeptide from the host cell.

[0025] In yet another aspect, the present invention provides the use of a polypeptide according to the present invention for the biotechnological production of levanbiose.

[0026] In still another aspect, the present invention provides a method for the production of levanbiose, comprising

[0027] (a) expressing a polypeptide according to the present invention in a suitable host cell; and

[0028] (b) contacting said polypeptide with a suitable substrate under conditions that allow the enzymatic production of levanbiose.

[0029] In some embodiments, said method of production is characterized in that the polypeptide is heterologously expressed in said host cell.

[0030] Alternatively or additionally, in some embodiments, said method is characterized in that the substrate comprises or consists of levan. In some embodiments, the method further comprises employment of at least one levan-degrading enzyme for degrading the substrate levan.

[0031] In some embodiments, said at least one levan-degrading enzyme is selected from the group of endo- levanases, preferably from the group of endo-levanases from Azotobacter chroococcum, preferably from the group of endo-levanases from A. chroococcum DSM 2286, and particularly the at least one levan- generating enzyme is LevB2286 from A. chroococcum DSM 2286.

[0032] Alternatively or additionally, in some embodiments, step (b) is carried out using a crude cell extract of the host cells of step (a).

[0033] Alternatively or additionally, in some embodiments, said host cell is selected from Escherichia coli strains.

[0034] Alternatively or additionally, in some embodiments, said method of production is characterized in that the concentration of hydrolysation side products other than levanbiose in the final product is less than 10 %, more preferably less than 7 % relative to the total amount of levanbiose obtained.

[0035] Alternatively or additionally, in some embodiments, said method is characterized in that the polypeptide of the present invention is expressed to have an activity yield of at least 60.000 U / l culture medium, measured as the amount of fructose equivalent generated from levan.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows activity yields obtained with different processes for production of levanbiose-generating enzymes. The activity is measured as the amount of fructose equivalent [pmol] generated from levan per minute and per litre microbial culture. Enzymes marked with a star (*) produce levanbiose with a purity of > 95 %.

[0038] Figure 2 shows a HPLC chromatogram of a solution containing both levan and the polypeptide having the amino acid sequence set forth in SEQ ID NO 1 after having been incubated for 2 hours (bold line). Solutions of fructose (F), levanbiose (DP2), and levantriose (DP3) were used as reference standards (dashed line). Glycerol (G), which was added to the purified enzyme to serve as a cryoprotectant, was introduced into the sample matrix.

[0039] Figure 3 shows SDS-PAGE (A) and volumetric activity yields (B) of crude cell extract obtained from E. coli NEB5-a pASK5_levMs (1), E. coli NEB5-a pASK5_levMs2(2), and E. coli NEB5-a pASK5_optRBS_levMs2(3). 10 pg of crude cell extracts were applied and visualized by fluorescent detection using a ChemiDoc Imager (Bio-Rad Laboratories Inc.). Volumetric activity yields were calculated based on the release of levanbiose [pmol per minute] and extrapolated to a culture volume of 1 L. The corresponding assays were carried out as biological duplicates. An ordinary one-way ANOVA was performed using GraphPad 10.2.3 (GraphPad Software, San Diego, US) to determine statistical significance; *: p = 0.0132; ****: p < 0.0001. Figure 4 shows viscosity of reaction mixtures set up for high-yield production of levanbiose. Varying concentrations of sucrose were converted to levan-based fructans using crude cell extract of E. coli DH5a pASK5_optRBS_levSi4i7_codon_optimized. Subsequently polymeric levan was hydrolyzed to levanbiose by LevMs2in the presence or absence of endo-levanase LevB2286. After enzymatic turnover, viscosities were assessed at a 20 ml scale using a Fungilab V-Pad Rotational Viscometer (R6 spindle, 250 rpm, 20 °C). An ordinary two-way ANOVA was performed using GraphPad 10.2.3 (GraphPad Software, San Diego, US) to determine statistical significance; ns = not significant; *: p = 0.0209; ****: p < 0.0010.

[0040] Figure 5 shows HPLC chromatograms of enzymatic reaction set up for high-yield production of levanbiose. Sample preparation and chromatographical analysis was performed according to Wienberg et al. (F. Wienberg, M. Hovels, K. Kosciow, U. Deppenmeier, High-resolution method for isocratic HPLC analysis of inulin-type fructooligosaccharides, J Chromatogr B Analyt Technol Biomed Life Sci 1172 (2021)). Enzymatic reactions were carried out as biological triplicates. Displayed are representative samples of enzyme reactions based on an initial sucrose concentration of 2 M before addition of enzymes, after the addition of recombinant levansucrase LevSi4i7, and after the addition of recombinant levanbiohydrolase LevMs2in the presence or absence of recombinant endo-levanase LevB2286. The enzymatic setup used allowed no separation of levanbiose and sucrose. Before the addition of LevMs2, sufficient sucrose consumption by LevSi4i7 was confirmed for each reaction. 6-kestose and 1-kestose are early stage fructosylation products accumulating during LevSi4i7 mediated levan-synthesis. Gly, glycerol; F, fructose; G, glucose; L / S, levanbiose and sucrose; 6-K, 6-kestose; 1-K; 1-kestose.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] Before describing in detail exemplary embodiments of the present invention, definitions which are important for understanding the present invention are given.

[0043] As used in this specification and in the appended claims, the singular forms of “a” and “an” also include the respective plurals unless the context clearly dictates otherwise.

[0044] “At least one”, as used herein, relates to one or more, in particular 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. “At least” if used in relation to a numerical value and in particular a list of numerical values relates to each of said separate numerical values and defines said numerical value as the minimum value. “At least 80, 81 , 82, etc.” thus means at least 80, at least 81 , at least 82 and so on.

[0045] In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10 %, preferably ±5 %, more preferably ±2 %.

[0046] The term “peptide” is used throughout the specification to designate a polymer of amino acid residues connected to each other by peptide bonds. The terms “protein” and “polypeptide” are used interchangeably throughout the specification to designate a polymer of amino acid residues connected to each other by peptide bonds. A protein or polypeptide according to the present invention has preferably more than 100 amino acid residues.

[0047] “Nucleic acid” as used herein includes all natural forms of nucleic acids, such as DNA and RNA. Preferably, the nucleic acid molecules of the invention are DNA.

[0048] Generally, the skilled person understands that for putting the present invention into practice any nucleotide sequence described herein may comprise an additional start and / or stop codon or that a start and / or stop codon included in any of the sequences described herein may be deleted, depending on the nucleic acid construct used. The skilled person will base this decision, e.g., on whether a nucleic acid sequence comprised in the nucleic acid molecule of the present invention is to be translated and / or is to be translated as a fusion protein. In various embodiments, the isolated polypeptides of the invention additionally comprise the amino acid M on the N-terminus.

[0049] “Isolated” as used herein in relation to a molecule means that said molecule has been at least partially separated from other molecules it naturally associates with or other cellular components. “Isolated” may mean that the molecule has been purified to separate it from other molecules and components, such as other proteins and nucleic acids and cellular debris, in particular those that accompany it due to its recombinant production in host cells.

[0050] Determination of the sequence identity of nucleic acid or amino acid sequences can be done by a sequence alignment based on well-established and commonly used BLAST algorithms (See, e.g. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403- 410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”; Nucleic Acids Res., 25, S.3389-3402). Such an alignment is based on aligning similar nucleotide or amino acid sequences stretches with each other. Another algorithm known in the art for said purpose is the FASTA algorithm. Alignments, in particular multiple sequence comparisons, are typically done by using computer programs. Commonly used are the Clustal series (See, e.g., Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31 , 3497-3500), T-Coffee (See, e.g., Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217) or programs based on these known programs or algorithms.

[0051] Also possible are sequence alignments using the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, USA) with the set standard parameters, with the AlignX module for sequence comparisons being based on the ClustalW. If not indicated otherwise, the sequence identity is determined using the BLAST algorithm.

[0052] Such a comparison also allows determination of the similarity of the compared sequences. Said similarity is typically expressed in percent identify, i.e., the portion of identical nucleotides / amino acids at the same or corresponding (in an alignment) sequence positions relative to the total number of the aligned nucleotides / amino acids. For example, if in an alignment 90 amino acids of a 100 aa long query sequence are identical to the amino acids in corresponding positions of a template sequence, the sequence identity is 90%. The broader term “homology” additionally considers conserved amino acid substitutions, i.e. amino acids that are similar in regard to their chemical properties, since those typically have similar chemical properties in a protein. Accordingly, such homology can be expressed in percent homology. If not indicated otherwise, sequence identity and sequence homology relate to the entire length of the aligned reference sequence, i.e., in the present case, e.g., SEQ ID NO: 3 or SEQ ID NO: 1. In some embodiments, the polypeptide of the present invention has, over its entire length, the sequence identity as herein defined. For instance, but without limitation, the polypeptide of the invention may be a shortened variant of SEQ ID NO: 3 that comprises at least 392 continuous amino acids of the amino acid sequence of SEQ ID NO: 3 (equaling 80 % sequence identity).

[0053] In the context ofthe present invention, a polypeptide of the present invention, i.e., a polypeptide comprising an amino acid sequence as herein defined, does not correspond to the amino acid sequence set forth in SEQ ID NO: 2; in other words, the amino acid sequence set forth in SEQ ID NO: 2, over its entire length, is not identical to a polypeptide of the present invention.

[0054] In the context of the present invention, the feature that an amino acid position corresponds to a numerically defined position in a reference sequence means that the respective position correlates to the numerically defined position in said reference sequence in an alignment obtained as described above.

[0055] It is to be understood that the term “comprising” is not limiting. For the purposes of the present invention the term “consisting of’ is considered to be a preferred embodiment ofthe term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

[0056] When referring to compositions and the weight percent of the therein comprised ingredients it is to be understood that according to the present invention the overall amount of ingredients does not exceed 100 % (± 1 % due to rounding).

[0057] It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0058] As surprisingly found by the present inventors, an N-terminally truncated variant of the wildtype parent levanbiohydrolase, said wildtype parent levanbiohydrolase having the amino acid sequence set forth in SEQ ID NO: 2, exhibits substantially improved levanbiose-generating activity relative to the wildtype parent set forth in SEQ ID NO: 2. Furthermore, as surprisingly found by the present inventors, a C-terminally truncated variant of said wildtype parent levanbiohydrolase (SEQ ID NO: 2) exhibits significantly improved expression rates relative to said wildtype parent. N-terminally truncated and optionally C-terminally truncated variants, as provided herein, therefore allow for economic enzymatic synthesis of levanbiose. These truncations resulted in increased product specificity towards levanbiose and significantly improved recombinant enzyme yields, as demonstrated for LevMs (N-terminal truncation only) and LevMs2(N- and C-terminal truncation).

[0059] Consequently, in a first aspect, the present invention relates to a polypeptide comprising an amino acid sequence having at least 70 %, such as at least 71 , 72, 73, 74, 75, 76, 77, 78, or 79 %, preferably at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, more preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, even more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal and, optionally, C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2, wherein each of N-terminal and C-terminal truncation is independently by at least one amino acid, such as at least two, three, four, five, six, seven, eight or nine amino acids, wherein said truncated polypeptide is a functional enzyme in that it has at least 80 %, such as at least 81 , 82, 83 or 84 %, preferably at least 85 %, such as at least 86, 87, 88 or 89 %, more preferably at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at least 95 %, such as at least 96, 97, 98 or 99 %, most preferably at least 100 %, such as 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 115, 120, 125, 130, 135, 140, 145 or 150 % of the activity, i.e., levanbiose-generating activity, of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 (under the same conditions).

[0060] Said functionality, i.e. that the claimed polypeptide has a certain percentage of levanbiose generating activity of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:2, can be determined by known and routinely applied tests for measuring enzyme activity. As a non-limiting example for measuring the activity of a levanbiose-generating enzyme, the following protocol may be applied: incubation of a levanbiose-producing enzyme with a substrate solution containing levan, preferably purified levan, at amounts in the range of 300 mM to 600 mM, such as in the range of 400 mM to 550 mM, preferably at an amount of about 500 mM levan, and a suitable buffer (such as Mcllvaine buffer (phosphate-citrate buffer) (pH of 5.5)); monitoring the increase in levanbiose concentration (for example by (discontinuous) sampling of the respective synthesis batch and subsequent HPLC analysis, as described in Hovels et al. (Hovels, M., Kosciow, K., Kniewel, J., Jakob, F., & Deppenmeier, U. (2020). High yield production of levan- type fructans by Gluconobacter japonicus LMG 1417. International Journal of Biological Macromolecules, 164, 295-303)).

[0061] In various embodiments, the N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, such as by at least 11 , 12, 13 or 14 amino acids, preferably at least 15 amino acids, such as at least 16, 17, 18, 19 or 20 amino acids, more preferably by at least 25 amino acids, such as at least 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids, but preferably by not more than 100 amino acids, such by at most 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72 or 71 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2. In some embodiments, the C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, such as by at least 11 , 12, 13 or 14 amino acids, preferably by at least 15 amino acids, such as by at least 16, 17, 18, 19 or 20 amino acids, more preferably by at least 25 amino acids, such as at least 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids, but preferably by not more than 100 amino acids, such by at most 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72 or 71 amino acids, most preferably by amino acids 553-620 of SEQ ID NO:2.

[0062] In various embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 70 %, preferably at least 80 %, more preferably at least 90 %, still more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0063] In various such embodiments, N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, but preferably by not more than 80 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0064] Alternatively or additionally, in various embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0065] Alternatively or additionally, in various embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0066] In various embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 70 %, preferably 80 %, more preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, obtainable by N-terminal and C- terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2, wherein each of N-terminal and C-terminal truncation is independently by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0067] In various such embodiments, N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, but preferably by not more than 80 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0068] Alternatively or additionally, C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, but preferably by not more than 80 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0069] Alternatively or additionally, in various embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation and C- terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2, as defined above, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0070] Alternatively or additionally, in various embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation and C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2, as defined above, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0071] Alternatively or additionally, in some embodiments, the polypeptide of the present invention is a polypeptide having at least 70 %, such as at least 71 , 72, 73, 74, 75, 76, 77, 78, or 79 %, preferably at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, more preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal and, optionally, C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2, wherein each of N-terminal and C-terminal truncation is independently by at least one amino acid, such as at least two, three, four, five, six, seven, eight or nine amino acids, wherein said truncated polypeptide has at least 80 %, such as at least 81 , 82, 83 or 84 %, preferably at least 85 %, such as at least 86, 87, 88 or 89 %, more preferably at least 90 %, such as at least 91 , 92, 93 or 94 %, particularly at least 95 %, such as at least 96, 97, 98 or 99 %, most preferably at least 100 %, such as 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 115, 120, 125, 130, 135, 140, 145 or 150 % of the activity, i.e., levanbiose generating activity, of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 (under the same conditions).

[0072] In the context of the present invention, “a polypeptide having a given sequence identity” relates to “a polypeptide consisting of an amino acid sequence having a given sequence identity”. For instance, but without limitation, “a polypeptide having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3” relates to “a polypeptide consisting of an amino acid sequence having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3”.

[0073] In various embodiments such embodiments, the N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, such as by at least 11 , 12, 13 or 14 amino acids, preferably at least 15 amino acids, such as at least 16, 17, 18, 19 or 20 amino acids, more preferably by at least 25 amino acids, such as at least 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids, but preferably by not more than 100 amino acids, such by at most 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72 or 71 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0074] In some other such embodiments, the C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, such as by at least 11 , 12, 13 or 14 amino acids, preferably by at least 15 amino acids, such as by at least 16, 17, 18, 19 or 20 amino acids, more preferably by at least 25 amino acids, such as at least 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids, but preferably by not more than 100 amino acids, such by at most 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72 or 71 amino acids, most preferably by amino acids 553-620 of SEQ ID NO:2.

[0075] In various further embodiments, the polypeptide of the present invention is a polypeptide having at least 70 %, preferably at least 80 %, more preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0076] In various such embodiments, N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, but preferably by not more than 80 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0077] Alternatively or additionally, in various embodiments, the polypeptide of the present invention is a polypeptide having at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0078] Alternatively or additionally, in various embodiments, the polypeptide of the present invention is a polypeptide having 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0079] In various embodiments, the polypeptide of the present invention is a polypeptide having at least 70 %, preferably 80 %, more preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, obtainable by N-terminal and C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2, wherein each of N-terminal and C-terminal truncation is independently by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0080] In various such embodiments, N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, but preferably by not more than 80 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0081] Alternatively or additionally, C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, but preferably by not more than 80 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0082] Alternatively or additionally, in various embodiments, the polypeptide of the present invention is a polypeptide having at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation and C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2, as defined above, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0083] Alternatively or additionally, in various embodiments, the polypeptide of the present invention is a polypeptide having 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal truncation and C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2, as defined above, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the levanbiose-generating activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0084] Alternatively or additionally, in various further embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1 .

[0085] In some embodiments, the polypeptide of the present invention is a polypeptide having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1.

[0086] It is furthermore contemplated that the polypeptides of the present invention may be modified, such as to facilitate purification, allow for detection, modify expression and / or secretion thereof, etc. The modifications may be of a minor nature, i.e., may be conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function.

[0087] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu, and Asp / Gly.

[0088] Alternatively or additionally, the polypeptides of the invention may comprise additional sequences that may include, among others, an affinity tag and / or a protease recognition and cleavage site as well as other proteins / polypeptides to which the polypeptides of the invention are fused, thus forming a fusion protein. Examples of such proteins to which the polypeptides of the invention may be fused include, without limitation, albumins and antibodies, as well as antibody fragments or antibody-like molecules and antibody derivatives.

[0089] The term “affinity tag” as used herein relates to entities which are coupled to a molecule of interest and allow enrichment of the complex between the molecule of interest and the affinity tag using an affinity tag receptor. In certain embodiments affinity tags may be selected from the group consisting of the Strep-tag® or Strep-tag® II, the myc-tag, the FLAG-tag, the His-tag, the small ubiquitin-like modifier (SUMO) tag, the covalent yet dissociable NorpD peptide (CYD) tag, the heavy chain of protein C (HPC) tag, the calmodulin binding peptide (CBP) tag, or the HA-tag or proteins such as Streptavidin binding protein (SBP), maltose binding protein (MBP), and glutathione-S-transferase. In some embodiments, a suitable tag may be selected from amino acid sequences having at least 85 %, such as at least 90 %, for instance 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0090] Therefore, in some embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0091] In various other embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 9.

[0092] In some embodiments, the polypeptide of the present invention may comprise a protease (recognition and) cleavage site. The term “protease (recognition and) cleavage site” refers to a peptide sequence which can be cleaved by a selected protease thus allowing the separation of peptide or protein sequences which are interconnected by a protease cleavage site. In certain embodiments the protease cleavage site is selected from the group consisting of a Factor Xa, a tobacco edge virus (TEV) protease, a enterokinase, a SUMO Express protease, an Arg-C proteinase, an Asp-N endopeptidases, an Asp-N endopeptidase + N-terminal Glu, a caspase 1 , a caspase 2, a caspase 3, a caspase 4, a caspase 5, a caspase 6, a caspase 7, a caspase 8, a caspase 9, a caspase 10, a chymotrypsin-high specificity, a chymotrypsin-low specificity, a clostripain (Clostridiopeptidase B), a glutamyl endopeptidase, a granzyme B, a pepsin, a prolineendopeptidase, a proteinase K, Welqut protease, Clean Cut protease, a staphylococcal peptidase I, a Thrombin, a Trypsin, intein, and a Thermolysin cleavage site. It can be preferred, in some embodiments, to design the protease recognition site such that as few amino acids as possible of the recognition and cleavage site remain attached to the peptide or protein of interest.

[0093] In various embodiments, the polypeptide may be derivatized or conjugated to another chemical moiety, said derivatization / conjugation including, amongst others, PEGylation, glycosylation. In particular, PEGylation, i.e., covalent coupling to polyethylene glycol of various molecularweights, is known as a means to alter pharmacokinetics of such compounds.

[0094] The polypeptide of the present invention may be an isolated polypeptide, as herein defined.

[0095] The invention further relates to the nucleic acid, in particular the isolated nucleic acid molecule, encoding the polypeptide of the present invention, as herein described above. In various embodiments, the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:5.

[0096] In some embodiments, the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6.

[0097] In some embodiments, the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:7.

[0098] In some embodiments, the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NQ:10.

[0099] In some embodiments, the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:11 .

[0100] Exemplary nucleic acid sequences encoding exemplary polypeptides of the present invention are set forth in the following Table 1 :

[0101] Table 1 : Amino acid sequences and corresponding nucleic acid sequences in accordance with the present invention

[0102] If the polypeptide comprises in addition to the amino acid sequence specified herein optional further amino acid sequences, all of these amino acid sequences are typically linked by peptide bonds and expressed as a single fusion protein. To facilitate said expression, the nucleic acid molecule comprises nucleotide sequences encoding all amino acid sequences, with said nucleotide sequences being operably linked to allow expression of the single fusion protein comprising all afore-mentioned amino acid sequences. Insofar not already present in the respective nucleic acid sequence encoding the polypeptide of the present invention, the nucleic acid sequence of the present invention may be operably linked to a start codon, for instance ATG, at the 5’ end of the nucleic acid sequence to allow for expression thereof.

[0103] Corresponding amino acid sequences of such a polypeptide according to the present invention, wherein the corresponding nucleic acid sequence comprises a start codon, may be defined as follows:

[0104] In some embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:16.

[0105] In some embodiments, the polypeptide of the present invention is a polypeptide having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:16.

[0106] In some embodiments, the polypeptide of the present invention is a polypeptide comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:17.

[0107] In some embodiments, the polypeptide of the present invention is a polypeptide having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:17.

[0108] The corresponding nucleic acid sequences encoding these polypeptides are defined as follows:

[0109] In some embodiments, the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:18.

[0110] In some embodiments, the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:19.

[0111] The term “operably linked” in the context of nucleic acid sequences means that a first nucleic acid sequence is linked to a second nucleic acid sequence such that the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter sequence is operably linked to a coding sequence of a heterologous gene if the promoter can initiate the transcription of the coding sequence. In a further context, a sequence encoding the polypeptide of the present invention is linked such to another amino acid sequence, that if the two sequences are translated a single peptide / protein chain is obtained.

[0112] In certain embodiments, the above defined nucleic acid molecules may be comprised in a vector, for example a cloning or expression vector. Generally, the nucleic acid molecules of the invention can also be part of a vector or any other kind of cloning vehicle, including, but not limited to a plasmid, a phagemid, a phage, a baculovirus, a cosmid, or an artificial chromosome. Generally, a nucleic acid molecule disclosed in this application may be “operably linked” to a regulatory sequence (or regulatory sequences) to allow expression of this nucleic acid molecule. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Non-limiting examples of regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.

[0113] Such cloning vehicles can include, besides the regulatory sequences described above and a nucleic acid sequence of the present invention, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art and are commercially available.

[0114] In certain embodiments the nucleic acid molecules disclosed herein are comprised in a cloning vector.

[0115] Therefore, in a further aspect, the present invention also relates to a vector comprising a nucleic acid molecule according to the present invention, wherein preferably the vector is a plasmid.

[0116] In some embodiments the nucleic acid molecules disclosed herein are comprised in an expression vector. The vectors may comprise regulatory elements for replication and selection markers. In certain embodiments, the selection marker may be selected from the group consisting of genes conferring ampicillin, kanamycin, chloramphenicol, tetracycline, blasticidin, spectinomycin, gentamicin, hygromycin, and zeocin resistance. In various other embodiments, the selection may be carried out using antibiotic-free systems, for example by using toxin / antitoxin systems, cer sequence, triclosan, auxotrophies or the like. Suitable methods are known to those skilled in the art.

[0117] The above-described nucleic acid molecule of the present invention, comprising a nucleic acid sequence encoding for the polypeptide of the invention, if integrated in a vector, must be integrated such that the polypeptide can be expressed. Therefore, a vector of the present invention comprises sequence elements which contain information regarding to transcriptional and / or translational regulation, and such sequences are “operably linked” to the nucleotide sequence encoding the polypeptide. An operable linkage in this context is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions comprise a promoter which, in prokaryotes, contains both the promoter per se, i.e., DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35 / - 10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'- capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.

[0118] In addition, the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.

[0119] In various embodiments, a vector comprising a nucleic acid molecule of the invention can therefore comprise a regulatory sequence, preferably a promoter sequence. In certain embodiments, the promoter is identical or homologous to promoter sequences of the host genome. In such cases endogenous polymerases may be capable to transcribe the nucleic acid molecule sequence comprised in the vector. In various embodiments, the promoter is selected from the group of weak, intermediate and strong promoters, preferably from weak to intermediate promoters.

[0120] In another preferred embodiment, a vector comprising a nucleic acid molecule of the present invention comprises a promoter sequence and a transcriptional termination sequence. Suitable promoters for prokaryotic expression are, for example, the araBAD promoter, the tet-promoter, the lacUV5 promoter, the CMV promotor, the EF1 alpha promotor, the AOX1 promotor, the tac promotor, the T7promoter, or the lac promotor. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter. Furthermore, a nucleic acid molecule of the invention can comprise transcriptional regulatory elements, e.g., repressor elements, which allow regulated transcription and translation of coding sequences comprised in the nucleic acid molecule. Repressor element may be selected from the group consisting of the Lac-, AraC-, or MalR-repressor.

[0121] In some embodiments, the nucleic acid molecule according to the present invention comprises a 5' untranslated region (5'-UTR) located upstream of the start codon of the nucleic acid sequence of the present invention. This means that the nucleic acid molecule of the present invention, defined as detailed herein above, is operably linked to a 5'-UTR. In some embodiments, the 5'-UTR comprises 3 to 30 nucleotides, such as 5 to 20 nucleotides, for instance 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides. In some embodiments, the 5'-UTR comprises or consists of a nucleic acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12.

[0122] In various embodiments, the nucleic acid molecule according to the present invention comprises a ribosome-binding site (RBS). This means that the nucleic acid molecule, as herein defined above, is operably linked to an RBS. Preferably, said RBS is located upstream of the start codon of the nucleic acid molecule of the present invention. In some embodiments, the nucleic acid molecule of the present invention is operably linked to a 5'-UTR and the RBS is comprised within said 5'-UTR. In some embodiments, an RBS suitable for employment in the context of the present invention comprises or consists of a nucleic acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, preferably SEQ ID NO: 13. Inclusion of such a preferred RBS, e.g., as set forth in SEQ ID NO: 13, may result in further optimization of recombinant expression of the nucleic acid molecule of the present invention.

[0123] The vector may be effective for prokaryotic or eukaryotic protein expression. Suitable vectors are known to those skilled in the art.

[0124] The vectors of the present invention may be chosen from the group consisting of high, medium and low copy vectors.

[0125] The above-described vectors of the present invention may be used for the transformation or transfection of a host cell in order to achieve expression of a peptide or protein which is encoded by an above-described nucleic acid molecule and comprised in the vector DNA.

[0126] Thus, in a further aspect, the present invention also relates to a host cell comprising a vector or nucleic acid molecule as disclosed herein.

[0127] Also contemplated herein are host cells, which comprise a nucleic acid molecule as described herein integrated into their genomes. The skilled person is aware of suitable methods for achieving the nucleic acid molecule integration. For example, the molecule may be delivered into the host cells by means of liposome transfer or viral infection and afterwards the nucleic acid molecule may be integrated into the host genome by means of homologous recombination. In certain embodiments, the nucleic acid molecule is integrated at a site in the host genome, which mediates transcription of the peptide or protein of the invention encoded by the nucleic acid molecule. In various embodiments, the nucleic acid molecule further comprises elements which mediate transcription of the nucleic acid molecule once the molecule is integrated into the host genome and / or which serve as selection markers.

[0128] In certain embodiments, the nucleic acid molecule of the present invention is transcribed by a polymerase natively encoded in the host genome. In various embodiments, the nucleic acid molecule is transcribed by an RNA-polymerase which is non-native to the host genome. In such embodiments, the nucleic acid molecule of the present invention may further comprise a sequence encoding for a polymerase and / or the host genome may be engineered or the host cell may be infected to comprise a nucleic acid sequence encoding for an exogenous polymerase. The host cell may be specifically chosen as a host cell capable of expressing the gene. In addition or otherwise, in order to produce the isolated polypeptide of the invention, the nucleic acid coding for it can be genetically engineered for expression in a suitable system. Transformation can be performed using standard techniques (Sambrook, J. et al. (2001), Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Prokaryotic or eukaryotic host organisms comprising such a vector for recombinant expression of the polypeptide as described herein form also part of the present invention. Suitable host cells can be prokaryotic cells. In certain embodiments the host cells are selected from the group consisting of gram positive and gram-negative bacteria. In some embodiments, the host cell is a gram-negative bacterium, such as Escherichia coli (E. coli). In certain embodiments, the host cell is E. coli. In further embodiments, the host cell is selected from the group consisting of E. coli, Pseudomonas, Serratia marcescens, Salmonella, Shigella (and other enterobacteriaceae), Neisseria, Hemophilus, Klebsiella, Proteus, Enterobacter, Helicobacter, Acinetobacter, Moraxella, Stenotrophomonas, Bdellovibrio, Vibrio, Legionella, acetic acid bacteria, Bacilli, Corynebacterium, Clostridium, Listeria, Streptococcus, Staphylococcus, and Archaea cells. Suitable eukaryotic host cells are among others CHO cells, insect cells, fungi, yeast cells, e.g., Saccharomyces cerevisiae, S. pombe, Pichia pastoris, and the like.

[0129] In some embodiments, the host cell is selected from the group consisting of E. coli, in particular E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coli XL) , E. coli NEB5-a, E. coli DH5a, E. coli DH1 , E. coli DM1 , E. coli HB101 , E. coli JmlOI-110, E. coli Rosetta(DE3)pLysS, E. co / / SURE, E. coli TOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue.

[0130] The transformed host cells are cultured under conditions suitable for expression of the nucleotide sequence encoding the polypeptide of the invention. In certain embodiments, the cells are cultured under conditions suitable for expression of the nucleotide sequence encoding a polypeptide of the invention and, optionally, its secretion.

[0131] For producing the polypeptide of the present invention, a vector of the invention can be introduced into a suitable prokaryotic or eukaryotic host organism by means of recombinant DNA technology (as already outlined above). For this purpose, the host cell is first transformed with a vector comprising a nucleic acid molecule according to the present invention using established standard methods (Sambrook, J. et al. (2001), supra). The host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or from the cultivation medium.

[0132] For expression of the polypeptides of the present invention several suitable protocols are known to the skilled person. The method for expression of a recombinant polypeptide of the present invention may be achieved by the following method comprising: (a) introducing a nucleic acid molecule or vector of the invention into a host cell, wherein the nucleic acid molecule or vector encodes the polypeptide of the present invention; and (b) cultivating the host cell in a culture medium under conditions that allow expression of the polypeptide of the present invention, and optionally secretion of the polypeptide into the culture medium.

[0133] Step (a) may be carried out by using suitable transformation and transfection techniques known to those skilled in the art. These techniques are usually selected based on the type of host cell into which the nucleic acid is to be introduced. In some embodiments, the transformation may be achieved using electroporation or heat shock treatment of the host cell. Step (b) may include a cultivation step that allows growth of the host cells. Alternatively, such step allowing growth of the host cells and a step that allows expression of the polypeptide may be performed separately in that the cells are first cultivated such that they grow to a desired density and then they are cultivated under conditions that allow expression of the polypeptide. The expression step can however still allow growth of the cells.

[0134] The method may further include a step of recovering the expressed polypeptide. The polypeptide may be recovered from the growth medium, if it is secreted, orfrom the cells or both. The recovery ofthe polypeptide may include various purification steps.

[0135] Generally, any known culture medium suitable for growth of the selected host may be employed in this method.

[0136] In various embodiments, the method also encompasses the purification of the polypeptide, wherein the polypeptide is purified using a method selected from affinity chromatography, ion exchange chromatography, reverse phase chromatography, size exclusion chromatography, and combinations thereof.

[0137] In several embodiments, the method may comprise the treatment ofthe polypeptide with a protease suitable for cleavage of a protease cleavage site within the polypeptide. In some embodiments, the polypeptide is purified prior to proteolytic cleavage using one or more methods disclosed above. Also after cleavage of peptide or protein, the method may comprise a further purification step as defined above. Thus, in some embodiments polypeptide is purified, subjected to proteolytic cleavage and the resulting polypeptide is further purified. In other embodiments, the protease may be co-expressed or added to the cultivation medium or expressed by co-cultivated microorganisms, such that cleavage occurs before purification.

[0138] In a further aspect, the present invention relates to the use of a vector or nucleic acid molecule as disclosed herein for the expression of a polypeptide according to the present invention. In some embodiments, the vector is used for the expression and optionally secretion of the polypeptide. The expression or expression and secretion may be achieved using the method described herein.

[0139] Therefore, in a further aspect, the present invention relates to a method for the production of a polypeptide of the present invention, comprising

[0140] (1) cultivating the host cell of the present invention under conditions that allow the expression of the polypeptide;

[0141] (2) optionally isolating the expressed polypeptide from the host cell.

[0142] In a further aspect, the present invention relates to the use of a polypeptide, as herein described and defined, for the biotechnological production of levanbiose.

[0143] A method for the production of levanbiose according to the present invention typically comprises the following steps: (a) expressing a polypeptide according to the present invention in a suitable host cell; and

[0144] (b) contacting said polypeptide with a suitable substrate under conditions that allow the enzymatic production of levanbiose.

[0145] In various embodiments, the method forthe production of levanbiose is characterized in that the polypeptide is heterologously expressed in said host cell.

[0146] In the context of the present invention, the term “suitable host cell” refers to a cell, for example a microbial cell, that is able to express a polypeptide of the present invention, as herein defined and described, preferably such that said polypeptide has an activity of at least 60.000, preferably at least 70.0000, more preferably at least 80.000 units per litre culture medium (unit = amount of fructose [pmol] generated per minute from levan).

[0147] Non-limiting examples of suitable host cells include, in particular, E. coli, such as E. coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coli XL1 , E. coli NEB5-a, E. coli DH5a, E. coli DH1 , E. coli DM1 , E. coli HB101 , E. coli JmlOI-110, E. coli Rosetta(DE3)pLysS, E. coli SURE, E. coli TOP10, E. coli XLI-Blue, E. coli XL2-Blue, and E. coli XLIO-Blue.

[0148] The polypeptide of the present invention may be expressed in said host cell, as herein defined, by means of an expression plasmid. Alternatively or additionally, integration of the levansucrase coding sequence into the host genome may occur.

[0149] In some embodiments, step a) includes a cultivation step. Depending on the type of host cell selected, the total culturing period may be in the range of about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35 or about 30 hours, preferably < 30 hours, such as about 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, or 17 hours, preferably < 17 hours, more preferably < 15 hours, even more preferably < 12 hours, still more preferably < 10 hours.

[0150] The culturing times indicated may vary depending on the type and / or volume of culturing. The cultivation step may include one or more preculturing steps and one or more main culturing steps. For instance, but without limitation, it may be necessary to carry out a culture cascade, particularly with regard to very large fermentation volumes, such that at least one step of preculturing and at least one step of main culturing may be required. Additionally or alternatively, longer preculturing and / or main culturing times might be required.

[0151] In some embodiments, step (b) is carried out using a crude cell extract of the host cells of step (a). Methods for obtaining a crude cell extract from a microbial culture are generally known in the art.

[0152] In some embodiments, in order to facilitate purification of the final product, i.e., levanbiose, and / or improve overall performance of the polypeptide according to the present invention, the crude cell extract may be obtained by lysis of the cells to obtain a lysed cell composition and removal of any solid components from the lysed cell composition to obtain the crude cell extract, such as by filtration or centrifugation and decantation.

[0153] In particular embodiments, the crude cell extract contains the polypeptide of the present invention, as herein defined and described, for example in such an amount that it exhibits a specific activity (Vmax) of at least 60.000, preferably at least 70.0000, more preferably at least 80.000 units per litre culture medium, such as an activity of 60.000, 65.000, 70.000, 75.000, 80.000, 85.000, 90.000, 95.000, 100.000, or 110.000 units per litre culture medium. The activity values reflect the maximum conversion rate Vmax, measured at physiological conditions (30 °C and pH 5,5) and a substrate concentration of at least 500 mM. The activity values were quantified by discontinuous sampling of a corresponding reaction mixture and subsequent HPLC analysis, as described in Hovels et al. (Hovels, M., Kosciow, K., Kniewel, J., Jakob, F., & Deppenmeier, U. (2020). High yield production of levan-type fructans by Gluconobacter japonicus LMG 1417. International Journal of Biological Macromolecules, 164, 295-303).

[0154] Due to such high enzymatic activity, the resultant crude cell extract can be used as is, i.e., without further purification of the polypeptide of the present invention.

[0155] In various embodiments, step (a) of the method of the present invention comprises the following steps: i) optionally preculturing the host cells in a suitable preculture medium; ii) culturing the host cells in a suitable main culture medium; and iii) obtaining a crude cell extract of the host cell culture of ii).

[0156] For instance, it may be advantageous if step (a) comprises the following steps: i) preculturing the host cells in a suitable preculture medium; ii) culturing the host cells in a suitable main culture medium; and iii) obtaining a crude cell extract of the host cell culture of ii).

[0157] For the generation of levanbiose, said crude cell extract is typically combined with a composition comprising a suitable substrate, e.g., levan, typically in form of an aqueous solution, allowing the polypeptide to catalyze hydrolysis of levan to levanbiose. In various embodiments, such substrate solutions may contain levan in an amount in the range of about 80 to about 200 g / L, such as about 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 g / L, preferably about 100 to about 180 g / L, more preferably about 140 to about 170 g / L sucrose, for instance about 170g / L.

[0158] For instance, but without limitation, said composition comprising levan, in addition to a base such as water, for instance tap water or deionized water, may comprise one or more additional components, such as, for instance but without limitation, buffers and stabilizing agents, antioxidants, etc. The pH of such a composition may be, for instance but without limitation, within the range of about 4.5 to 10, such as about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, preferably within the range of about 4.5 to 8, such as about 5 to 6, such as, for instance, between about 5.25 to 5.75. Thus, in various embodiments, the substrate comprises or consists of levan.

[0159] For instance, but without limitation, it may be advantageous from an economical point of view if the substrate comprises or consists of a levan-containing solution produced using a levan-generating enzyme, e.g., a levansucrase used to generate levan from sucrose. In such embodiments, the levan-containing solution may contain other types of fructooligosaccharides generated by the levan-generating enzyme.

[0160] One major advantage of the method for production of levanbiose according to the present invention resides in the fact that the polypeptide of the present invention generates levanbiose with a high degree of purity. Therefore, in particular embodiments, the production method of the present invention is characterized in that the concentration of hydrolysis side products other than levanbiose in the final product is less than 10 %, such as less than 9 % or less than 8 %, more preferably less than 7 %, such as about 6, 5, 4, 3, 2 or 1 % or less, relative to the total amount of levanbiose obtained.

[0161] Particularly in cases where a levan-containing solution produced using a levan-generating enzyme is used as the substrate solution, purification of levanbiose may be performed. Levanbiose can be isolated and purified by means generally known in the art, such as in a preparatory way using, for example, chromatography on activated carbon powder and / or ion-exchange chromatography.

[0162] While such an enzymatic cascade for levanbiose production - comprising enzymatic levan formation by a levan-generating enzyme, such as, without limitation, LevSi4i7 from Gluconobacter japonicus LMG 1417 and subsequent levan hydrolysis in accordance with the present invention, such as by relying on a polypeptide of SEQ ID NO: 3 (LevMs2) - enables high product yields, it may be challenging at industrial scale, since the intermediate formation of high-molecular-weight levan may lead to strongly elevated viscosities, particularly at high sucrose concentrations. At levels above ~5% (w / v), polymeric levan typically exhibits gel-like behavior due to molecular entanglement, thus potentially impairing membrane filtration (flux decline, fouling), spray drying (poor atomization), mixing and / or pumping (high energy demand).

[0163] To mitigate this viscosity-related limitation, the present inventors developed an optimized approach combining a polypeptide of the present invention with ultra-low doses of at least one endo-levanase. This approach leverages minimal endo-hydrolytic activity to fragment high-molecular-weight levan polymers, thereby reducing molecular entanglement without affecting product selectivity (cf. Example 3). As found, employment of at least one endo-levanase sufficiently reduces the intrinsic viscosity compared to reactions in which only a polypeptide of the present invention was added for levan hydrolysis, while retaining high levanbiose yield and purity regardless of presence or absence of the endo-levanase. This system is a scalable and selective process for the production of levanbiose, particularly from highly concentrated and viscous levan-solutions, even at ultra-low dosages of the endo-levanase.

[0164] Consequently, in various embodiments, the method for production of levanbiose according to the present invention comprises employment of at least one levan-degrading enzyme for degrading polymeric levan as the substrate for the polypeptide of the present invention so as to decrease the viscosity of the substrate solution, or overall reaction mixture. The term “levan-degrading enzyme” refers to an enzyme capable of degrading polymeric levan that is different from the polypeptide of the present invention. In various embodiments, said at least one levan-degrading enzyme is selected from the group of endo-levanases, such as endo-levanases from Bacteroides thetaiotaomicron (for instance BT1760 from Bacteroides thetaiotaomicron DSM 2079), Bacillus licheniformis (such as LevB1 from Bacteroides thetaiotaomicron DSM 13), or Azotobacterchroococcum (for instance LevB2286 from Azotobacter chroococcum), as disclosed in WO 2020 / 260249 A1 , preferably from the group of endo-levanases from Azotobacter chroococcum, preferably from the group of endo-levanases from A. chroococcum DSM 2286, particularly the at least one levan-generating enzyme is LevB2286 from A. chroococcum DSM 2286. In various embodiments, the at least one levan-degrading enzyme comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In other embodiments, the at least one levan-degrading enzyme comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. Preferably, the at least one levan-degrading enzyme comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0165] In order to effectively decrease viscosity of the reaction mixture, employment of low doses of levantdegrading enzyme are sufficient. In some embodiments, the ratio of the at least one levan-degrading enzyme to the polypeptide of the present invention is from about 1 : 50 to about 1 : 2000, more preferably from about 1 :100 to about 1 :1500, still more preferably from about 1 :150 to about 1 :1000, particularly from about 1 :200 to about 1 :9000, more particularly from about 1 :300 to about 1 :800, such as about 1 :350, 1 :400, 1 :450, 1 :500, 1 :550, 1 :600, 1 :650, 1 :700, 1 :750, 1 :800, 1 :850 or 1 :900.

[0166] In some embodiments, both the at least one levan-degrading enzyme and the polypeptide of the present invention is in the form of a crude cell extract, wherein the ratio of the volume of crude cell extract containing the at least one levan-degrading enzyme to volume of crude cell extract containing the polypeptide of the present invention is from about 1 : 50 to about 1 : 2000, more preferably from about 1 :100 to about 1 :1500, still more preferably from about 1 :150 to about 1 :1000, particularly from about 1 :200 to about 1 :9000, more particularly from about 1 :300 to about 1 :800, such as about 1 :350, 1 :400, 1 :450, 1 :500, 1 :550, 1 :600, 1 :650, 1 :700, 1 :750, 1 :800, 1 :850 or 1 :900.

[0167] Generally, employment of the at least one levan-degrading enzyme, as herein described and defined, results in a decrease in viscosity of the levan-containing substrate solution or levan-containing reaction mixture. Accordingly, this particular variation of the production method of the present invention is particularly suitable for employment with levels of polymeric levan of at least about 2 % (w / v) in the substrate composition, preferably with levels of polymeric levan of at least about 3, 4 or 5 % (w / v), particularly of at least about 5 % (w / v). In some embodiments, employment of the at least one levan-degrading enzyme is combined with levels levan in the levan-containing substrate solution of about 2 % (w / v) to about 20 % (w / v), such as about 3, 4, 5, 6, 7, 8, 9,10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 % (w / v).

[0168] Furthermore, generally, employment of the at least one levan-degrading enzyme, as herein described and defined, does not result in a decrease of yields of levanbiose relative to a method of production of levanbiose according to the present invention that does not comprise employment of the at least one levandegrading enzyme. In various embodiments, said “decrease of yields of levanbiose” refers to a decrease by 50 % or more, 45 % or more, 40 % or more, 35 % or more, 30 % or more, 25 % or more, 20 % or more, 15 % or more, 10 % or more or 5 % or more, relative to a method of production of levanbiose according to the present invention that does not comprise employment of the at least one levan-degrading enzyme.

[0169] Alternatively or additionally, in some embodiments, the method of producing levanbiose is characterized in that the polypeptide is expressed to have an activity yield of at least 60.000 U / l culture medium, preferably at least 65.000 U / l culture medium, more preferably at least 70.000 U / l culture medium, most preferably at least 75.000 U / l culture medium, such as at least 80.000 U / l culture medium, measured as the amount of fructose equivalents [pmol] generated per minute from levan.

[0170] Analysis of the product formed in accordance with the present invention can be carried out for example by HPLC inverse phase on a Nucleosil C18 column, using for instance a mixture of water and methanol as the eluent, and / or NMR after isolation. Alternatively or additionally, analysis of the product formed can be carried out by HILIC-chromatography, employing an amino (NH2) column, using for instance a mixture of water and acetonitrile as the eluent.

[0171] In the following, particular embodiments of the present invention are described:

[0172] Embodiment 1 : Polypeptide comprising an amino acid sequence having at least 70 %, preferably at least 80 %, more preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal and, optionally, C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0173] Embodiment 2: Polypeptide according to embodiment 1 , wherein the N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0174] Embodiment 3: Polypeptide according to embodiment 1 or embodiment 2, wherein the C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, most preferably by amino acids 553-620 of SEQ ID NO:2.

[0175] Embodiment 4: Polypeptide according to any one of embodiments 1 to 3, comprising an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1 . Embodiment 5: Polypeptide according to embodiment 1 , having at least 70 %, preferably at least

[0176] 80 %, more preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N-terminal and, optionally, C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the activity of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0177] Embodiment 6: Polypeptide according to embodiment 5, wherein the N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

[0178] Embodiment 7: Polypeptide according to embodiment 5 or embodiment 6, wherein the C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, most preferably by amino acids 553-620 of SEQ ID NO:2.

[0179] Embodiment 8: Polypeptide according to any one of embodiments 5 to 7, having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1.

[0180] Embodiment 9: Nucleic acid encoding the polypeptide of any one of embodiments 1 to 8, wherein optionally:

[0181] - the nucleic acid sequence comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:5; or

[0182] - the nucleic acid sequence comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6; or

[0183] - the nucleic acid sequence comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:7; or

[0184] - the nucleic acid sequence comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NQ:10; or

[0185] - the nucleic acid sequence comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:11 ; or the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:18; or

[0186] - the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 86, 87, 88 or 89 %, preferably at least 90 %, such as at least 91 , 92, 93, 94, 95, 96, 97, 98 or 99 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO:19.

[0187] Embodiment 10: Nucleic acid encoding the polypeptide of any one of embodiments 1 to 9, wherein the nucleic acid molecule is operably linked to a 5'-UTR, wherein optionally the 5 -UTR comprises 3 to 30 nucleotides, such as 5 to 20 nucleotides, for instance 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides and / or the 5'-UTR comprises or consists of a nucleic acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12.

[0188] Embodiment 11 : Nucleic acid encoding the polypeptide of any one of embodiments 1 to 10, wherein the nucleic acid molecule is operably linked to an RBS. Preferably, said RBS being located upstream of the start codon of the nucleic acid molecule, wherein optionally the RBS is comprised within a 5 -UTR operably linked to the nucleic acid molecule and / or the RBS comprises or consists of a nucleic acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 12 or in SEQ ID NO: 13, preferably SEQ ID NO: 13.

[0189] Embodiment 12: Vector comprising a nucleic acid molecule according to any one or embodiments

[0190] 9 to 11 , wherein the vector preferably is a plasmid.

[0191] Embodiment 13: Vector according to embodiment 12, wherein the vector is the plasmid pASK5.

[0192] Embodiment 14: Host cell comprising a nucleic acid molecule according to embodiment 9 or a vector according to embodiment 10, the host cell preferably being a prokaryotic host cell.

[0193] Embodiment 15: Host cell according to embodiment 14, wherein the host cell is selected from E. coli, optionally from genetically engineered E. coli, optionally selected from E. coli NEB5-a and E. coli DH5a.

[0194] Embodiment 16: Method for the production of a polypeptide of any one of embodiments 1 to 8, comprising

[0195] (1) cultivating the host cell of embodiment 11 under conditions that allow the expression of the polypeptide;

[0196] (2) optionally isolating the expressed polypeptide from the host cell.

[0197] Embodiment 17: Use of a polypeptide according to any one of embodiments 1 to 8 for the biotechnological production of levanbiose.

[0198] Embodiment 18: Method for the production of levanbiose, comprising

[0199] (a) expressing a polypeptide according to any one of embodiments 1 to 8 in a suitable host cell; and

[0200] (b) contacting said polypeptide with a suitable substrate under conditions that allow the enzymatic production of levanbiose.

[0201] Embodiment 19: The method according to embodiment 18, wherein the polypeptide is heterologously expressed in said host cell; and / or the substrate comprises or consists of levan; and / or step (b) is carried out using a crude cell extract of the host cells of step (a); and / or the host cell is Escherichia coli.

[0202] Embodiment 20: The method according to embodiment 18 or embodiment 19, wherein the concentration of hydrolysation side products other than levanbiose in the final product is less than 10 %, more preferably less than 7 % relative to the total amount of levanbiose obtained; and / or the polypeptide is expressed to have an activity yield of at least 60.000 U / l culture medium, measured as the amount of fructose equivalents [pmol] generated per minute from levan.

[0203] Embodiment 21 : The method according to any one of embodiments 18 to 20, further comprising employment of at least one levan-degrading enzyme for degrading the substrate levan, wherein preferably the ratio of the at least one levan-degrading enzyme to the polypeptide according to any one of embodiments 1 to 8 is from about 1 : 50 to about 1 : 2000, more preferably from about 1 :100 to about 1 :1500, still more preferably from about 1 :150 to about 1 :1000, particularly from about 1 :200 to about 1 :9000, more particularly from about 1 :300 to about 1 :800, such as about 1 :350, 1 :400, 1 :450, 1 :500, 1 :550, 1 :600, 1 :650, 1 :700, 1 :750, 1 :800, 1 :850 or 1 :900.

[0204] Embodiment 22: The method according to embodiment 21 , wherein both the at least one levandegrading enzyme and the polypeptide according to any one of embodiments 1 to 8 is in the form of a crude cell extract, wherein the ratio of the volume of crude cell extract containing the at least one levan-degrading enzyme to volume of crude cell extract containing the polypeptide according to any one of embodiments 1 to 8 is from about 1 : 50 to about 1 : 2000, more preferably from about 1 :100 to about 1 :1500, still more preferably from about 1 :150 to about 1 :1000, particularly from about 1 :200 to about 1 :9000, more particularly from about 1 :300 to about 1 :800, such as about 1 :350, 1 :400, 1 :450, 1 :500, 1 :550, 1 :600, 1 :650, 1 :700, 1 :750, 1 :800, 1 :850 or 1 :900.

[0205] Embodiment 23: The method according to embodiment 21 or embodiment 22, wherein employment of the at least one levan-degrading enzyme results in a decrease in viscosity of the substrate solution or reaction mixture. Embodiment 24: The method according to any one of embodiments 21 to 23, wherein the level of polymeric levan in the substrate composition is at least about 2 % (w / v), preferably at least about 3, 4 or 5 % (w / v), particularly at least about 5 % (w / v).

[0206] Embodiment 25: The method according to any one of embodiments 21 to 24, wherein employment of the at least one levan-degrading enzyme does not result in a decrease in yields of levanbiose relative to a method of production of levanbiose in accordance embodiment 18 that does not comprise employment of the at least one levan-degrading enzyme.

[0207] Embodiment 26: The method according to any one of embodiments 21 to 25, wherein said at least one levan-degrading enzyme is selected from the group of endo-levanases, preferably from the group of endo-levanases from Azotobacter chroococcum, preferably from the group of endo-levanases from A chroococcum DSM 2286, and particularly the at least one levan-generating enzyme is LevB2286 from A chroococcum DSM 2286.

[0208] Embodiment 27: The method according to any one of embodiments 21 to 26, wherein the at least one levan-degrading enzyme comprises or consists of an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

[0209] In the following, non-limiting examples that illustrate various aspects of the present invention are described.

[0210] Examples

[0211] Example 1 : Recombinant production of the polypeptide according to the present invention and enzymatic synthesis of levanbiose using the same

[0212] Recombinant production of the polypeptide encoded by SEQ ID NO:1 (N-terminal truncation, LevMs) orthe C-terminally truncated version of the polypeptide encoded by SEQ ID NO:1 , as set forth in SEQ ID NO:3 (LevMs2) was routinely carried out as described in the following protocols. The protocols also contain relevant information for the downstream enzymatic synthesis of levanbiose using the recombinant polypeptides encoded by SEQ ID NO:1 and SEQ ID NO:3. The following exemplary protocols are based on a genetically modified Escherichia coli DH5a strain harboring the expression plasmid pASK5_ / ev / V?s (used for the production of the polypeptide encoded by SEQ ID NO:1).

[0213] Preculture

[0214] Material from a cryo-conserved stock of a genetically engineered Escherichia coli DH5a strain harboring the expression plasmid pASK5_ / ev / V?s (used for expression of SEQ ID NO:1) was transferred into a sterile shake flask filled with an appropriate growth medium (e.g. LB-Miller medium). To facilitate plasmid maintenance, the culture was supplemented with the antibiotic carben icilli n at a concentration of 100 pg / ml. Cultivation was carried out for 10 hours in a shaking incubator at 250 rpm and 37 °C.

[0215] Main culture

[0216] Sterile Enhanced 2x YT medium (20 g / l yeast extract, 32 g / l tryptone, 10 g / l NaCI, 2.5 g / l N32HPO4, and 2 g / l glucose, pH 7.4) was supplemented with the antibiotic carbenicillin at a concentration of 100 pg / ml. The medium was transferred into a sterile shake flask and inoculated with 5 % [v / v] of the respective preculture of E. coli DH5a pASK5_ / ev / V?s. Cultivation was carried out in a shaking incubator at 250 rpm and 37 °C. After reaching an optical density (measured at 600 nm) of 1 , expression of the recombinant gene was induced by adding anhydrotetracycline at a concentration of 0.2 pg / ml. After induction, the main culture was incubated for 16 hours at 250 rpm and 18 °C.

[0217] Preparation of crude cell extract

[0218] Biomass generated during the cultivation of the main culture was pelleted by centrifugation (13,000 RCF, 10 minutes, 4 °C) using an appropriate centrifuge (e. g. Beckman Avanti JXN-26). The resulting cell pellet was resuspended in cold Buffer W (100 mM Tris, 150 mM NaCI, pH 8) using 5 ml buffer per g wet biomass. For cell lysis, the cell suspension was subjected to sonification using an appropriate sonicator (e. g. Bandelin Sonopuls HD 2070.2 equipped with Bandelin Sonotrode VS 70 T). Under constant cooling, the suspension was exposed to an amplitude of 50 % for two minutes per ml of cell suspension and at a pulse interval of 50 %. Afterward, cell debris was removed by centrifugation (20,000 RCF, 15 minutes, 4 °C) and subsequent filtration using a syringe filter (PVDF, 0.2 pm). For storage, the resulting crude cell extract was mixed with an equal volume of sterile glycerol and transferred to -20 °C.

[0219] Enzymatic synthesis of levanbiose

[0220] For levanbiose-synthesis, the produced crude cell extract of E. coli DH5a pASK5_ / ev / V?s was incubated with a substrate solution containing elevated amounts of levan. A suitable substrate solution may contain pure levan, purified after enzymatic synthesis from sucrose using a suitable purification process (e.g., dialysis) and Mcllvaine buffer with a pH of 5.5. Another suitable substrate for the obtained crude cell extract may be a levan-containing process solution produced from sucrose using a suitable levansucrase (e.g., the levansucrase LevSi4i7 from Gluconobacterjaponicus LMG 1417). Said process solution may preferably be adjusted to pH 5.5 using Mcllvaine buffer.

[0221] The crude cell extract obtained from a 1 -liter main culture of E. coli DH5a pASK5_ / ev / V?s contains sufficient amounts of the polypeptide encoded by SEQ ID NO:1 to convert 200 Liter of a high concentrated levan solution (e.g., 500 mM). The increase in levanbiose concentration can be monitored by discontinuous sampling of the respective synthesis batch and subsequent HPLC analysis as described in Hovels et al. (Hovels, M., Kosciow, K., Kniewel, J., Jakob, F., & Deppenmeier, U. (2020). High yield production of levan- type fructans by Gluconobacter japonicus LMG 1417. International Journal of Biological Macromolecules, 164, 295-303).

[0222] Example 2: Optimization of recombinant expression

[0223] Analogous to Example 1 , the influence of presence of an optimized RBS upstream of the start codon on the recombinant production of the amino acid molecule LevMs2(N- and C-terminal truncation; SEQ ID NO: 3), was evaluated using genetically engineered Escherichia coli NEB5-a strain harboring the expression plasmid pASK5_levMs2or pASK5_optRBS_levMs2:

[0224] Table 2: 5’-UTR of the gene levMs2encoded on the plasmids pASK5_levMs2and pASK5_optRBS_levMs2The variable regions upstream of the ATG start codon are highlighted in bold.

[0225] The modification resulted in a previously unattained volumetric activity yield of 357,615 ± 45,431 U / L culture in E. coli NEB5-a pASK5_optRBS_levMs2(Fig. 3). One unit (U) is defined as the amount of levanbiose [pmol] released per minute from polymeric levan. This represents a >25-fold increase over the original construct (13,050 ± 471 U / L for E. coli NEB5-a pASK5_levMs) and a 3.5-fold increase over the non-RBS- optimized LevMs2variant (103,495 ± 2,913 U / L for E. coli NEB5-a pASK5_levMs2). Importantly, the high specificity of levanbiose production was retained across all constructs, with final product purities consistently exceeding 95 % [w / w]. These results constitute a valuable extension, demonstrating that targeted RBS engineering enables industrial-scale LevMs2production while preserving product selectivity.

[0226] Example 3: Optimization of production process viscosities

[0227] While the enzymatic cascade for levanbiose production - comprising for example levan formation by LevSi4i7 from Gluconobacter japonicus LMG 1417 and subsequent levan hydrolysis by LevMs2- enables high product yields, it introduces a significant process engineering challenge at industrial scale. The intermediate formation of high-molecular-weight levan leads to strongly elevated viscosities, particularly at high sucrose concentrations. At levels above ~5% (w / v), polymeric levan exhibits gel-like behavior due to molecular entanglement, severely impairing membrane filtration (flux decline, fouling), spray drying (poor atomization), and mixing or pumping (high energy demand). Starting from intrinsic viscosities of 14.95 (1 M sucrose), 25.44 (1.5 M sucrose), and 33.74 (2 M Sucrose) mPa*s the addition of LevSi4i7 led to a profound increase in viscosity reaching a maximum of 484.6 ± 72.9 mPa*s (Table 3). Subsequent levan hydrolysis by LevMs2effectively degraded the polymeric levan to the extent that no precipitable levan remained. However, a residually increased viscosity persisted (Table 3), particularly at higher sucrose concentrations, suggesting that incomplete disruption of transient polymer networks continued to affect the rheological properties of the system.

[0228] Table 3: Viscosity of enzyme reactions comprising LevS-u -mediated synthesis of polymeric levan and subsequent hydrolysis by LevMs2. Substrate solutions containing defined concentrations of sucrose were first converted to polymeric levan using recombinant LevSi4i7- Reactions were heat-inactivated and subsequently supplemented with recombinant LevMS2. At the end of the enzymatic reactions, intrinsic viscosities were assessed using a Fungilab V- Pad Rotational Viscometer (R6 spindle, 250 rpm, 20 °C).

[0229] To mitigate this viscosity-related limitation, a modified approach combining LevMs2with ultra-low doses of the endo-levanase LevB2286 from Azotobacter chroococcum DSM 2286 (M. Hovels, K. Kosciow, U. Deppenmeier, Characterization of a novel endo-levanase from Azotobacter chroococcum DSM 2286 and its application for the production of prebiotic fructooligosaccharides, Carbohydr Polym 255 (2021)) was developed:

[0230] Recombinant production of levan-forming and degrading enzymes

[0231] Cultivation of E. coli strains expressing truncated LevMs2from M. laevaniformans ATCC 15953, the levansucrase LevSi4i7 from G. japonicus LMG 1417, or the endo-levanase LevB2286 from A. chroococcum DSM 2286 (M. Hovels, K. Kosciow, U. Deppenmeier, Characterization of a novel endo-levanase from Azotobacter chroococcum DSM 2286 and its application for the production of prebiotic fructooligosaccharides, Carbohydr Polym 255 (2021); U. Deppenmeier, M. Hovels, K. Kosciow, Enzymatic production of levan-based, prebiotic fructooligosaccharides, 2022) was performed in Enhanced 2xYT medium (20 g L-1yeast extract, 32 g / L-1tryptone / peptone from casein, 10 g L-1NaCI, 2 g L-1glucose, 3.06 g L-1Na2HPO4) adjusted to pH 7.4. To avoid the formation of Maillard products, glucose was autoclaved separately. After sterilization, carbenicillin was added to all cultures at a final concentration of 100 pg mL-1to maintain plasmid stability. Precultures, inoculated from cryopreserved stocks were incubated overnight at a 25 mL scale in 250 mL Erlenmeyer flasks at 250 rpm and 37 °C. The main cultures, carried out at a 250 mL scale in 2.5 L Erlenmeyer flasks, were inoculated with 12.5 ml of the respective preculture and incubated at 250 rpm and 37 °C. At an optical density (600 nm) of 1 , expression was induced by adding 25 pL of anhydrotetracycline stock solution (2 mg mL-1). After induction, the cultures were maintained at 250 rpm and 18 °C for 16 hours. Afterward, cells were harvested by centrifugation (15 min, 8,000xg, 4 °C) and resuspended in 5 ml of chilled buffer W (100 mM Tris-HCI, 150 mM NaCI, 1 mM EDTA, pH 8.0) per g wet biomass. For cell lysis, the suspensions were subjected to ultrasonication using a Branson Sonifier Cell Disruptor B15 (Thermo Fisher Scientific, Waltham, US). Sonication was carried out for 2 min mL-1at an output intensity of 7 and a pulse interval of 50 %. Cleared lysates were obtained by centrifugation (15 min, 15,000xg, 4 °C) of the sonicated suspensions, sterile filtered (0.22 pm, PVDF), mixed with equal volumes of sterile glycerol and stored at - 20 °C.

[0232] Quantification of volumetric activity yields of LevM-producing E. coll strains

[0233] The activity of crude cell extracts containing the truncated LevM variants LevMs (N-terminal truncation, SEQ ID NO: 1) and LevMs2(N- and C-terminal truncation, SEQ ID NO: 3) was assessed by HPLC-assisted quantification of levanbiose, released from Timothy grass levan. One unit of activity was defined as the amount of levanbiose (pmol) released per minute per volumetric unit. Results were extrapolated to calculate activity yields per liter of bacterial culture. All assays were performed as biological duplicates and periodically sampled for subsequent HPLC analysis. Chromatographic analysis was performed based on the HPLC setup established by Wienberg et al. (F. Wienberg, M. Hovels, K. Kosciow, U. Deppenmeier, High-resolution method for isocratic HPLC analysis of inulin-type fructooligosaccharides, J Chromatogr B Analyt Technol Biomed Life Sci 1172 (2021)), which enabled the rapid analysis of inulin-based FOS up to a DP of 17. Assays supplemented with crude cell extract were sampled periodically and analyzed using a SpectraSYSTEM HPLC-system (Thermo Fisher Scientific Inc., Waltham, US) equipped with a degasser (SpectraSYSTEM SCM1000), a pump (SpectraSYSTEM P4000), an autosampler (SpectraSYSTEM AS3000) and a refraction index (Rl) detector (RI-101 , Ercatech AG, Bern, Switzerland). Compound separation was achieved using the main column Asahipak NH2P-50 4E, the guard column Asahipak NH2P- 50G 4A, and an Asahipak NH2P-LF line filter (all Showa Denko Europe GmbH, Munich, Germany). The guard- and main column were heated to 40 °C. The mobile phase, 65 % [v / v] acetonitrile (MeCN), was applied in isocratic mode at a flow rate of 0.6 mL min-1. During enzyme assays, 50 pL samples were periodically withdrawn, mixed with 50 pL of H2Odemin, and supplemented with 400 pL of 96 % ethanol. Precipitated levan was removed by centrifugation (13,000xg, 1 min). Subsequently, 100 pL of the obtained supernatant was diluted with 300 pL 80 % MeCN. A final centrifugation was carried out for one minute at 13,000xg to remove insoluble matter before samples were subjected to HPLC analysis. An appropriate sample loop ensured a consistent injection volume of 20 pL. Detected compounds were quantified by the external standard method, as described by Wienberg et al..

[0234] Assessment of product profiles and viscosities during targeted levanbiose-synthesis

[0235] The impact of varying sucrose concentrations and enzyme combinations on the product profile and viscosity during targeted levanbiose-synthesis was validated. Therefore, at a 20 mL scale, sterile substrate solutions were set up containing 1 , 1 .5, or 2 M sucrose and 40 mM acetate buffer (pH 5.6). Before the reaction were supplemented with ideal amounts of crude E. coll extract containing the recombinant levansucrase LevS1417 (Table 3) intrinsic viscosities were assessed using a Fungilab V-Pad Rotational Viscometer (R6 spindle, 250 rpm, 20 °C).

[0236] Table 3: Volumetric data on LevS1417-mediated levan synthesis. Reactions were carried out at a 20 mL scale and incubated for 96 hours at 30 °C.

[0237] Initiated reactions were incubated at 30 °C for 96 hours and stopped by thermal inactivation. Therefore, all reactions were heated to 70 °C for 20 minutes. Sucrose turnover was assessed by HPLC and was considered successful if at least 90 % of the sucrose was converted (compared to a negative control supplemented with the corresponding volume of demin). The impact of levan formation on intrinsic viscosities was again assessed by a Fungilab V-Pad Rotational Viscometer (R6 spindle, 250 rpm, 20 °C). For levanbiose-synthesis, the reactions were supplemented with separate crude cell extracts containing recombinant LevMs2and LevB2286 (Table 4) and incubated for 18 hours at 30 °C.

[0238] Table 4: Volumetric data on targeted levanbiose synthesis catalyzed by the levanbiohydrolase LevMs2in the presence or absence of endo-levanase LevB2286. Levanbiose synthesis was performed at a 20 mL scale and incubated for 18 h at 30 °C.

[0239] Assays were thermally inactivated at 70 °C for 20 minutes and analyzes using a Fungilab V-Pad Rotational Viscometer (R6 spindle, 250 rpm, 20 °C). Product profiles were assessed by HPLC.

[0240] The described optimized approach leverages minimal endo-hydrolytic activity to fragment high-molecular- weight levan polymers, thereby reducing molecular entanglement without affecting product selectivity. In reactions containing initial sucrose concentrations > 1 .5 M, the addition of a minimal LevB2286 dose (1 pL of LevB2286-containing crude cell extract per 20 mL reaction volume) led to a statistically significant viscosity reduction (Fig. 4). On average, the use of LevB2286 reduced the intrinsic viscosity by 61 .1 ± 1.7 % compared to reactions in which only LevMs2was added for levan hydrolysis. The high levanbiose yield and purity (>95 % [w / w]) caused by the predominant activity of LevMs2was retained in all reactions regardless of the presence or absence of LevB2286 (Fig. 5). These findings validate the bi-enzymatic system (LevMs2+ LevB2286) as a scalable and selective process for the production of levanbiose from highly concentrated and viscous levan-solutions. In addition, these findings demonstrate the effectiveness of LevB2286 to resolve levan-related viscosity even at ultra-low dosage.

Claims

CLAIMS1. Polypeptide comprising an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:3, obtainable by N- terminal and, optionally, C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 by at least one amino acid, wherein said truncated polypeptide has at least 80 %, preferably at least 85 %, more preferably at least 90 %, particularly at least 95 %, most preferably at least 100 % of the activity, particularly levanbiose-generating activity, of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

2. The polypeptide according to claim 1 , wherein the N-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, most preferably by amino acids 1-64 of SEQ ID NO:2.

3. The polypeptide according to claim 1 or claim 2, wherein the C-terminal truncation of the polypeptide having the amino acid sequence set forth in SEQ ID NO:2 is by at least 10 amino acids, preferably at least 15 amino acids, more preferably by at least 25 amino acids, most preferably by amino acids 553-620 of SEQ ID NO:2.

4. The polypeptide according to any one of claims 1 to 3, comprising an amino acid sequence having at least 80 %, preferably at least 90 %, more preferably 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1 .

5. Nucleic acid encoding the polypeptide of any one of claims 1 to 4.

6. Vector comprising a nucleic acid molecule according to claim 5, wherein the vector preferably is a plasmid.

7. Host cell comprising a nucleic acid molecule according to claim 5 or a vector according to claim 6, the host cell preferably being a prokaryotic host cell.

8. Method for the production of a polypeptide of any one of claims 1 to 4, comprising(1) cultivating the host cell of claim 6 under conditions that allow the expression of the polypeptide;(2) optionally isolating the expressed polypeptide from the host cell.

9. Use of a polypeptide according to any one of the preceding claims for the biotechnological production of levanbiose.

10. Method for the production of levanbiose, comprising(a) expressing a polypeptide according to any one of claims 1 to 4 in a suitable host cell; and(b) contacting said polypeptide with a suitable substrate under conditions that allow the enzymatic production of levanbiose.

11. The method according to claim 10, wherein the polypeptide is heterologously expressed in said host cell; and / or the substrate comprises or consists of levan; and / or - step (b) is carried out using a crude cell extract of the host cells of step (a); and / or the host cell is Escherichia coli.

12. The method according to claim 10 or claim 11 , wherein the concentration of hydrolysation side products other than levanbiose in the final product is less than 10 %, more preferably less than 7 % relative to the total amount of levanbiose obtained; and / or the polypeptide is expressed to have an activity yield of at least 60.000 U / l culture medium, measured as the amount of fructose equivalent generated from levan.

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